Oxidative injury to the pulmonary endothelium plays an important role in ARDS. We have postulated that a strategy for specific targeting for oxidant-regulating enzymes to defined compartments of pulmonary endothelium is essential to understand and treat disease with an endothelial oxidation component, such as ARDS. The data we have accumulated over the last decade show that antibodies against endothelial surface antigens provide specific targeting of either oxidant-generating (glucose oxidase, GOX) or oxidant-detoxifying (SOD and catalase) enzymes to the pulmonary vascular endothelium in animals. The present project will focus on the use of antibodies against Platelet-Endothelial Cell Adhesion Molecule-1, anti-PECAM. Chemical modification of anti-PECAM with biotin and streptavidin provides a previously unrecognized ability for extremely effective pulmonary targeting of the conjugates and enables intracellular delivery of the enzymes, for either intracellular generation of detoxification of oxidants. We postulate that this latter property will be critical for effective protection against oxidants generated by or diffusing into endothelium. Based on our results, we hypothesize that: A) streptavidin-mediated conjugation of oxidant-regulating enzymes to anti- PECAM provides a novel mechanism for endothelium-specific intracellular pulmonary targeting of the enzymes; B) Targeting of an oxidant-producing enzyme, glucose oxidase, will lead to intraendothelial generation of H202 and produce intracellular oxidative stress in endothelial cells; C) Targeting of an oxidant-degrading enzyme, catalase, will provide a more specific and effective mechanism for protective against oxidant injury than administration of non-targeted antioxidants. This proposal will test these hypotheses via accomplishment of the following specific aims. 1. Study and optimize the intraendothelial targeting of enzymes conjugated to antibodies to PECAM-1. We will define the cellular destination and fate of the streptavidin/anti-PECAM conjugates, evaluate the mechanism of streptavidin-induced stimulation of pulmonary uptake and evaluate the mechanisms of streptavidin-induced facilitation of internalization of anti-PECAM. 2. Study endothelial oxidative stress induced by intracellular immunotargeting of the H202-generating enzyme, glucose oxidase (GOX). We will characterize intracellular generation of H202 by the endothelium- associated anti-PECAM/catalase versus non-targeted catalase. We will compare protection against intracellular and extracellular oxidants produced by different mechanisms (including anti-PECAM/GOX) in cell culture, in perfused rat lungs and in anesthetized animals. The anticipated results will provide a novel strategy for the specific manipulations of oxidants in the pulmonary vasculature and are important for further progress into basic mechanisms of endothelial oxidative stress.
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